Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression

Ethosuximide ameliorates C. elegans dnj-14 mutant phenotypes

The rare hereditary human neurodegenerative disease, autosomal-dominant adult-onset
neuronal ceroid lipofuscinosis (ANCL), is caused by mutations in the DNAJC5 gene 19]–22]. DNAJC5 encodes a neuronal chaperone of the DnaJ/Hsp40 family of molecular chaperones known
as cysteine string protein (CSP), which prevents the misfolding of presynaptic proteins
23]–27]. DNJ-14 is the worm orthologue of CSP and dnj-14 null mutants are characterised by reduced lifespan and age-dependent sensorimotor
defects and neurodegeneration, similar to CSP knockout mice 18], 28]. We used this dnj-14 model to screen for compounds with therapeutic potential for ANCL and possibly other
neurodegenerative diseases, by testing their ability to extend the short lifespan
of dnj-14(ok237) worms 18]. The anti-epileptic drug, ethosuximide, was observed to produce a robust and reproducible
lifespan extension in dnj-14(ok237) animals. This effect was concentration-dependent, with 1 mg/ml ethosuximide offering
the most significant lifespan increase, raising the mean lifespan of dnj-14(ok237) worms by over 40 % (Fig. 1a) Over a series of experiments, this optimal concentration produced a near-complete
rescue of lifespan in dnj-14 mutants to levels close to that of wild-type N2 worms
(Additional file 1: Table S1). At the highest concentration used (4 mg/ml), ethosuximide produced no
significant increase in lifespan. Notably, none of the concentrations used had any
significant effect on the lifespan of wild-type N2 C. elegans (Additional file 2: Figure S1A; Additional file 1: Table S1). To test if ethosuximide was also able to rescue the sensory defect in
dnj-14 mutants, we performed a food race assay, which measures the time taken for animals
to move a defined distance to a bacterial food source (Fig. 1b). As previously observed 18], dnj-14 mutants are severely impaired in this assay. Ethosuximide significantly improved
food sensing activity of dnj-14 mutants, approximately doubling the number of worms reaching the food within 60 min,
although complete rescue to wild type levels was not achieved (Fig. 1b). Ethosuximide had no stimulatory activity in food race assays using wild type N2
worms (Fig. 1b), nor did it increase locomotion of dnj-14 or N2 worms in thrashing assays (Additional file 2: Figure S1B). Therefore the stimulatory action of ethosuximide in food race assays
appears to be due to a specific effect on the chemosensory defect in dnj-14 mutants rather than a generic stimulation of movement. Taken together, these data
suggest that ethosuximide is able to ameliorate the neurotoxicity induced by the loss
of the DNJ-14 synaptic chaperone protein.

Fig. 1. Ethosuximide increases lifespan and improves sensorimotor function in C. elegans ANCL and frontotemporal dementia models.a Ethosuximide extends lifespan in dnj-14 mutants. Viability of age-synchronised dnj-14(ok237) animals grown in the presence of the indicated concentrations of ethosuximide was
determined; untreated wild type control N2 worms are shown for comparison (n?=?50-55
worms for each concentration). b Ethosuximide ameliorates the dnj-14 food sensing defect. The time taken to move to a bacterial food source was measured
in wild type N2 and dnj-14(tm3223) strains grown until 5-6 days of age in the presence or absence of ethosuximide (n?=?71-80
worms of each strain per condition). c Ethosuximide increases locomotion in Tau V337M worms, but not control worms. Thrashing
in solution was measured in Tau V337M worms grown until 1 and 3 days of age and assayed
in the presence of the indicated concentrations of ethosuximide (for each age group,
n?=?120-140 worms for 0 mg/l; n?=?38-40 worms for 0.1, 0.2 and 0.5 mg/ml; n?=?80-90
worms for 1 and 2 mg/ml;). Identically treated wild type control CZ1200 worms are
shown for comparison (n?=?20 worms per concentration). Data are shown as mean?±?SEM
(***p??0.001). d Age-dependence of ethosuximide’s effect on Tau V337M locomotion. Thrashing assays
were performed on age-synchronised animals grown in the presence or absence of 2 mg/ml
ethosuximide (n?=?30-50 worms per data point). Data are shown as mean?±?SEM (***p??0.001, *p??0.05). e Ethosuximide increases lifespan in Tau V337M worms. Viability of age-synchronised
animals grown in the presence of the indicated concentrations of ethosuximide was
determined in comparison to untreated wild type control CZ1200 worms (n?=?50-102 worms
for each drug concentration). f Comparison of the ethosuximide concentration-dependence of mean lifespan extension
in dnj-14 and Tau V337M worms

Ethosuximide alleviates phenotypes caused by expression of human mutant Tau

To determine if ethosuximide had general neuroprotective activity, we evaluated its
effects on a C. elegans frontotemporal dementia with parkinsonism-17 (FTDP-17) tauopathy model 29]. FTDP-17 is one of many human tauopathies in which characteristic neurofibrillary
tangles are formed from hyperphosphorylated Tau. Overexpression of human mutant Tau
V337M throughout the C. elegans nervous system causes severe motility defects, neurodegeneration, short lifespan
and accumulation of insoluble Tau 29]. The highly penetrant and easily observable motility phenotype of this model is well
suited for assessing drug effects 10]. We observed that ethosuximide improved the severely uncoordinated phenotype of Tau
V337M transgenic worms in solution. To quantify this effect, we performed thrashing
assays in the presence of varying concentrations of ethosuximide. As shown in Fig. 1c, ethosuximide increased the thrashing frequency of young worms in a dose-dependent
manner, with an optimal concentration of 1-2 mg/ml. Although this stimulatory effect
was highly significant (P??0.001) and approximately doubled thrashing rates at these concentrations, this
still represents only a relatively small increase that is far from a complete rescue
to wild type levels. Nevertheless, the effects were Tau-specific, because control
worms showed wild-type thrashing activity that was not increased by ethosuximide (Fig. 1c; Additional file 2: Figure S1C). Ethosuximide significantly increased thrashing in young animals (day
1 and 3), but its therapeutic activity declined in older animals (Fig. 1d). To directly test the effect of ethosuximide on longevity, lifespan assays were
performed on ethosuximide treated Tau V337M and wild type worms and compared with
vehicle-treated controls. Ethosuximide significantly enhanced the mean lifespan of
Tau V337M worms in a concentration-dependent manner (Fig. 1e; Additional file 1: Table S1), but had no effect on wild type control worms (Additional file 2: Figure S1D; Additional file 1: Table S1). Maximal lifespan extension was seen at 2 mg/ml, which conferred a 40 %
lifespan increase – comparable to the longevity effect of ethosuximide seen with dnj-14 mutants – although wild type lifespan was not significantly affected at any concentration
(Fig. 1f).

Ethosuximide action is independent of T-type calcium channels

The efficacy of ethosuximide in generalised absence epilepsy is thought to be due
to blockade of the low voltage activated T-type calcium channel 30]. C. elegans CCA-1 is most similar to the vertebrate T-type calcium channel ?1 subunit (42 % identity),
with typical T-type kinetics, voltage dependence and pharmacology 31]. We therefore constructed a double mutant Tau V337M; cca-1(ad1650) strain to determine if ethosuximide’s therapeutic action in the frontotemporal dementia
model was mediated via inhibition of CCA-1. We chose to use the Tau model because
it is extremely challenging technically to cross cca-1 with dnj-14 mutants, as both genes are located on the X chromosome. As seen in Fig. 2a, loss of cca-1 had minimal effect on Tau proteotoxicity, as a similar large percentage of both Tau
V337M; cca-1(ad1650) homozygotes and Tau V337M transgenic worms exhibited severely impaired motility.
In contrast, both cca-1(ad1650) single mutant control and heterozygous cross progeny did not exhibit any motility
defects. Ethosuximide treatment mitigated the impaired motility of Tau V337M transgenic
worms and double mutants harboring a loss-of-function mutation of cca-1 to a similar extent, both at day 1 and day 3 (Fig. 2a). Tau V337M; cca-1(ad1650) double mutants and Tau V337M transgenic worms displayed a mean adult lifespan of
12.1 and 12.6 days, respectively. Following ethosuximide supplementation, there was
negligible change in lifespan of cca-1(ad1650) single mutant control worms. However, the ability of ethosuximide to increase longevity
in the single Tau V337M transgenic strain was maintained in the Tau V337M; cca-1(ad1650) homozygotes, significantly extending the mean lifespan by 25 % and 16 %, respectively
(Fig. 2b). Taken together, these results indicate that the mechanism of ethosuximide action
does not involve inhibition of CCA-1.

Fig. 2. Ethosuximide acts independently of T-type calcium channels and bacterial metabolism
to reduce Tau aggregation. a Ethosuximide Inhibition of the T-type calcium channel, CCA-1, is not required for
protection against paralysis. Tau V337M transgenic animals were crossed with loss-of-function
mutations for cca-1(ad1650) to generate homozygous cross progeny. Ethosuximide supplementation increased thrashing
activity of the Tau V337M transgenic strain and the double mutant Tau V337M; cca-1(ad1650) strain to similar extents. Data are shown as mean?±?SEM (**p??0.01, *p??0.05; n?=?30-50 worms per data point). b Ethosuximide extends lifespan in Tau V337M mutants in the absence of CCA-1. Lifespan
assays were performed on single mutant cca-1(ad1650), transgenic (Tau V337M) and double mutant Tau V337M; cca-1(ad1650) strains grown in the presence (dashed lines) or absence (solid lines) of 1 mg/ml
ethosuximide (n??100 worms per strain/condition). c Ethosuximide extends lifespan using killed bacteria as a food source. Lifespan assays
were performed on Tau V337M worms grown under control conditions or in the presence
of kanamycin, or 1 mg/ml ethosuximide or both (n??80 worms per condition). d Total Tau protein expression in Tau V337M worm lysates is not reduced by ethosuximide.
The left panel shows a representative western blot; the right panel shows quantification
of Tau normalised to actin and expressed as % of untreated control (mean?±?SEM, n?=?3;
not significant). e, f Ethosuximide affects Tau proteostasis. e shows a representative western blot of the soluble and detergent-soluble (RIPA) sequentially
extracted fractions in the presence or absence of ethosuximide treatment. f shows quantification of Tau fractions normalised to actin and expressed as % of the
total (soluble?+?RIPA) protein level (data are shown as mean?±?SEM (n?=?3; *p??0.05)

Protective effects of ethosuximide are not due to changes in the E. coli food source

One contributor to late-age mortality in C. elegans is the detrimental effect of their E. coli food source, and drugs that decrease bacterial pathogenicity extend worm lifespan
32]. In addition, it has recently been shown that some drugs, for example metformin,
increase C. elegans lifespan indirectly via changes to E. coli metabolism 33]. To determine if such bacterial effects contribute to the mechanism of action of
ethosuximide in lifespan extension, we used the antibiotic kanamycin to kill the OP50
food source and thus prevent both bacterial metabolism and pathogenicity. In single
treatments, kanamycin and ethosuximide increased the mean lifespan of Tau V337M worms
by 37 % and 48 %, respectively (Fig. 2c). A combined treatment of kanamycin and ethosuximide caused a significant and approximately
additive 72 % extension in mean lifespan. As kanamycin clearly did not affect the
ability of ethosuximide to increase lifespan (Fig. 2c; Additional file 1: Table S1), this suggests that ethosuximide’s protective effects are independent
of bacterial metabolism and pathogenicity.

Ethosuximide affects Tau protein solubility

In view of the general association between protein misfolding/aggregation and neurodegeneration,
we reasoned that ethosuximide’s therapeutic activity might be linked to improved proteostasis.
We therefore tested whether ethosuximide could influence the aggregation of mutant
Tau protein in the worm frontotemporal dementia model. As shown in Fig. 2d, there was no reduction in total Tau levels in ethosuximide treated Tau transgenic
worms as compared to vehicle controls. The rescuing effect of ethosuximide is therefore
not due to transgene suppression or reduced expression of toxic mutant Tau protein.
We then subjected both vehicle- and ethosuximide-treated Tau V33M transgenic worms
to a regimen of sequential extractions with buffers of increasing solubilising strengths,
as previously described 29]. Quantification of the amount of soluble and insoluble (RIPA-extractable) Tau relative
to total Tau levels revealed a significant reduction in aberrantly-folded, insoluble
Tau and a corresponding increase in soluble Tau in ethosuximide-treated compared with
untreated worms (Fig. 2e,f). Therefore, ethosuximide’s protective effects on motility and longevity are accompanied
by improved Tau proteostasis.

Ethosuximide modulates expression of DAF-16 target genes

To gain insight into ethosuximide’s mechanism of action, we took an unbiased transcriptomic
approach using whole genome C. elegans DNA microarrays. Two control strains (N2 and CZ1200) and two ANCL model strains (dnj-14 ok237 and tm3223 alleles) were age-synchronised and treated with 1 mg/ml ethosuximide or vehicle control.
Gene expression profiling was then used to identify ethosuximide-responsive differentially
expressed genes (DEGs) in 6-day-old animals (Additional file 3: Figure S2). Principal component analysis confirmed tight grouping of the triplicate
biological samples for each strain and a consistent effect of ethosuximide, as illustrated
in the array correlation heatmap (Additional file 4: Figure S3A-C). In order to stringently select for the most consistent and significant
ethosuximide-regulated transcripts, we focused on DEGs common to at least 3 out of
4 strains using a 1 % false discovery rate (FDR) cut-off. This yielded 125 DEGs, comprising
61 up-regulated and 64 down-regulated genes. Restricting our analysis still further
to transcriptional changes common to all four strains yielded 60 DEGs containing 40
up-regulated and 20 down-regulated genes, as illustrated in Additional file 4: Figure S3D,E. The complete data are shown in Additional file 5: Dataset S1.

DEGs that showed at least a 2-fold change in all strains were considered the most
significant ethosuximide-responsive genes and are listed in Table 1. Given the known neuroprotective effect of inhibiting the DAF-2 insulin/IGF signalling
(IIS) pathway, it was notable that the upregulated genes included four members of
the dod (downstream of daf-16) gene class associated with the DAF-2 signalling pathway; asm-3, a regulator of the DAF-2 pathway; and ttr-44, which is upregulated in long-lived daf-2 mutants. The most strongly down-regulated transcripts were either uncharacterised
genes or individual genes associated with diverse cellular processes, for example
ubiquitination (fbxb-66). Seven of the up-regulated genes identified in our microarray experiments were chosen
for validation using qRT-PCR. Significantly increased expression by qRT-PCR was observed
for all 7 genes, thus confirming the microarray data (Fig. 3a). In contrast, qRT-PCR analysis showed no change in the expression level of pph-6 (chosen as a negative control based on our microarray data) or of two normalising
genes: pmp-3 and act-1.

Table 1. Gene expression changes induced by ethosuximide in all microarray experiments

Fig. 3. Ethosuximide-induced genes are enriched in DAF-16-associated elements and ethosuximide-induced
lifespan extension requires daf-16. a Validation of gene expression changes using qRT-PCR. Selected genes that were up-regulated
by ethosuximide in microarray experiments (ugt-25, dhs-26, cyp-14A3, cyp-35B1, ttr-44, dod-6 and cyp-34A9) were confirmed to be significantly induced using qRT-PCR. No significant changes
in expression of normalisation (act-1, pmp-3) or negative control (pph-6) genes was observed. Results are expressed as mean fold change?±?SEM relative to
the unexposed control (n?=?3). b Ethosuximide-responsive genes are enriched for the DAF-16 Associated Element (DAE)
motif. To identify regulatory sequences correlating with ethosuximide-responsiveness,
200-bp regions in the upstream promoter sequences of common DEGs were mined for overrepresented
motifs using RSAT. c Ethosuximide increases dnj-14 lifespan in a daf-16-dependent manner. Survival curves of dnj-14(ok237) worms grown on E. coli containing empty vector (L4440), hsp-1 or daf-16 dsRNA-producing plasmids in the presence (dashed lines) or absence (solid lines)
of 1 mg/ml ethosuximide. Ethosuximide treatment significantly increased the lifespan
of dnj-14(ok237) worms on vector control (p??0.001), but had no significant effect on daf-16 RNAi animals (p??0.15) (n??100 worms per strain/condition)

Functional annotation enrichment analysis of DEGs shared by at least 3 treated strains
was used to subdivide the ethosuximide-responsive transcripts into groups based on
Gene Ontology (GO) identifiers (Additional file 6: Figure S4). Both DAVID and modMine GO analyses yielded a significant enrichment
for GO terms related to “lipid glycosylation”, “lipid modification”, “oxidation reduction”,
“determination of adult lifespan” and “chromatin assembly”. DEGs up-regulated in response
to ethosuximide exposure clustered into four groups with significant enrichment scores
(Additional file 6: Figure S4A). Cluster 1 contained eight cytochrome P450 genes (cyp-14A3, cyp-34A2, cyp-34A9/dod-16, cyp-35B1/dod-13, cyp-35C1, cyp-33A1, cyp-35A2,
cyp-35A3), three short chain dehydrogenase genes (dhs-23, dhs-26, dhs-2) and an aldehyde dehydrogenase (alh-5). These enriched genes (Additional file 7: Figure S5) share annotation terms relating to oxidoreductase activity, ion binding
and multicellular organismal ageing. Clusters 2 and 3 are overlapping and contain
the same group of 6 putative UDP-glucuronosyl/glucosyl transferases (UGTs) involved
in lipid and phase II metabolism (F08A8.2, ugt-51, ugt-8, ugt-41, ugt-14, ugt-25). Cluster 4 is associated with the term ageing and lifespan determination and comprised
cyp-34A9, cyp-35B1, dod-6, dod-3, and ftn-1, which are amongst the most responsive downstream targets of DAF-16/FOXO; and thn-1 and spp.-1. DEGs down-regulated in response to ethosuximide showed a significant enrichment
to chromatin remodelling and related functional categories encompassing “cellular
macromolecular complex assembly, chromatin assembly or disassembly, DNA packaging,
nucleosome assembly, chromatin organisation, and chromatin assembly” (Additional file
6: Figure S4). Enriched genes (Additional file 7: Figure S5) include an H2B histone (his-8) and H1 linker histone variants (his-24, hil-2, hil-3, hil-7) which play roles in heterochromatin packaging and gene regulation. modMine publication
enrichment analysis (Additional file 6: Figure S4C) further revealed a significant enrichment for DAF-16 target genes.

Analysis of the upstream promoter regions of ethosuximide-responsive DEGs using Regulatory
Sequence Analysis Tools (RSAT) revealed that the most significantly enriched motif
was CTTATCA (Fig. 3b). This is the consensus sequence for the DAF-16-associated element (DAE), which is
overrepresented in the promoters of DAF-16-regulated target genes downstream of DAF-2
in the IIS pathway 34]. RSAT also identified accessory motifs that co-occurred with DAE, which appear to
be core promoter sequence elements in C. elegans (Additional file 8: Figure S6). No additional motifs were identified using 3 other tools (MEME-DREME,
SCOPE, BioProspector) and indeed DAE and its variants was the only regulatory sequence
identified by all four sequence analysis tools (Additional file 8: Figure S6).

Ethosuximide’s protective effect requires DAF-16

Subjecting C. elegans to mild stress can increase longevity via hormesis. However, comparing our microarray
data with the literature (Additional file 9: Figure S7), it is evident that the transcriptomic effect of ethosuximide was inconsistent
with a general stress response. Furthermore, ethosuximide did not induce oxidative
stress and the consequent transcriptional induction of SKN-1 target genes, as evidenced
by microarray data (Additional file 9: Figure S7), confirmatory qRT-PCR (Additional file 10: Figure S8A) and the lack of activation of a GFP reporter of the SKN-1 target, gst-4 (Additional file 10: Figure S8B). Finally, paraquat and juglone applied at concentrations that have previously
been shown to result in hormesis-induced lifespan extension did not rescue the dnj-14 mutant despite inducing strong Pgst-4::GFP expression (Additional file 10: Figure S8C).

As our microarray data had revealed DAF-16 target gene modulation as a major consequence
of ethosuximide application, we set out to test if DAF-16 was required for ethosuximide-mediated
protection. We were unable to obtain stable lines of homozygous double mutants for
both dnj-14(ok237) and daf-16(mu86), as the putative double mutants exhibited severe developmental problems and extremely
low brood size (Additional file 11: Figure S9), suggesting a synthetic lethal genetic interaction. We therefore used
RNAi to knockdown expression of daf-16, which resulted in a significant reduction in lifespan of both wild type and dnj-14(ok237) worms (Fig. 3c; Additional file 12: Figure S10). Ethosuximide treatment of dnj-14(ok237) worms on L4440 vector control RNAi bacteria caused a robust lifespan extension; but,
strikingly, daf-16 RNAi abolished this effect (Fig. 3c). RNAi of hsp-1, which encodes an Hsp70 protein, also reduced lifespan, but this was significantly
increased by ethosuximide (Additional file 12: Figure S10), indicating both that ethosuximide acts independently of hsp-1 and that inhibition by daf-16 RNAi is not a general consequence of RNAi. These data therefore suggest that DAF-16
is essential for the therapeutic action of ethosuximide.

To investigate if ethosuximide affects the subcellular localisation of DAF-16, we
examined the effect of the drug on a strain containing a DAF-16-GFP reporter (Additional
file 13: Figure S11). Although we could observe nuclear translocation of DAF-16-GFP in response
to heat shock and starvation, we were unable to detect an obvious effect of ethosuximide.
It is important to note, however, that this does not affect the conclusion that ethosuximide’s
action requires DAF-16 activity. For example, the classical IIS mutant age-1(hx546), which absolutely requires DAF-16 for lifespan extension, also does not exhibit increased
nuclear DAF-16-GFP 35]. It is possible that the effect of ethosuximide on nuclear enrichment of DAF-16-GFP
or the amount of nuclear DAF-16/FOXO required for the effects on lifespan might simply
be below the detection threshold in this type of experiment.

Ethosuximide modulates mammalian FOXO target gene expression

Given that DAF-16/FOXO is conserved between C. elegans and mammals, we tested whether ethosuximide could affect the transcriptional activity
of mammalian DAF-16 homologues (FOXO1, FOXO3, and FOXO4). FOXOs have been shown to
modulate cell cycle arrest, apoptosis, autophagy, angiogenesis, differentiation, stress
resistance, insulin signalling, stem cell maintenance and metabolism 36]. Nine classical mammalian FOXO target genes involved in cell cycle regulation (Ccng2, Cdkn1a, Cdkn1b, Rbl2), DNA repair (Gadd45a), apoptosis (Bim), stress response (Cat, Sod2) and insulin signalling (Eif4ebp1) were selected and their mRNA levels in a differentiated mouse neuroblastoma cell
line (N2A) following ethosuximide exposure were then measured using qRT-PCR. There
was no significant change in gene expression at 0.1 mg/ml, but at 0.56 mg/ml (the
optimal concentration that stimulates neuronal differentiation in stem cells 37]) and 1 mg/ml (the optimal concentration in our worm ND models) ethosuximide significantly
up-regulated the mRNA expression of FOXO target genes involved in cell cycle regulation
(Ccng2, Cdkn1b, Rbl2) and DNA damage repair (Gadd45a) (Fig. 4a). Therefore, the ability of ethosuximide to modulate DAF-16/FOXO target gene expression
is conserved from worms to mammals.

Fig. 4. Ethosuximide induces FOXO target gene expression and reduces polyglutamine protein
aggregation in mammalian neurons. a mRNA levels of classical FOXO target genes were analysed by qRT-PCR in differentiated
mouse N2A neuroblastoma cells treated with the indicated concentrations of ethosuximide.
Data are shown as mean?±?SEM (n?=?3; *p???0.05, **p???0.01). (b–c) Ethosuximide reduces polyglutamine protein aggregation. b Visualisation of EGFP-tagged non-pathological (polyQ25) and pathological (polyQ97)
polyglutamine tracts in N2A cells 72 h post-transfection. Phase contrast, GFP (green)
and SYTOX orange staining (red, to identify dead cells) confocal images are shown
to illustrate that aggregates are specific to polyQ97 and that ethosuximide redistributed
polyQ97-EGFP away from aggregates into the cytoplasm and neuronal processes C) Quantification
of polyQ aggregation. The number of polyQ-EGFP transfected N2A cells bearing fluorescent
aggregates as a percentage of the total number of viable transfected (green) cells
was counted at the indicated post-transfection times. Cell/aggregate counting was
performed manually and confirmed using ImageJ software if cells were sufficiently
sparse to allow this. Data are shown as mean?±?SEM (n?=?3, counting ~100 cells in
each experiment; *p??0.05)

Ethosuximide suppresses protein aggregation in mammalian neurons

To determine if ethosuximide could also affect proteostasis in mammalian neurons,
we transfected N2A cells with EGFP-tagged polyglutamine (polyQ) constructs and monitored
aggregate formation over time in culture 38]. PolyQ25 barely aggregated either in the presence or absence of ethosuximide, as
GFP staining was evenly distributed throughout the cytosol (Fig. 4b). In contrast, there was a progressive increase in the number of cells with polyQ97
aggregates, with intracellular GFP punctae readily observable in the cytoplasm of
most polyQ97-transfected N2A cells after 72 h (Fig. 4b, Additional file 14: Figure S12). When aggregates appeared, cells tended to round up and GFP-fluorescence
in the cytosol was restricted to bright punctae. Ethosuximide treatment appeared to
antagonise aggregate formation and to increase diffuse cytosolic GFP staining in the
soma and neuronal processes, suggesting that it enhanced the solubility of polyQ97.
To quantify this, individual GFP aggregates were counted and the percentage of polyQ-EGFP
transfected N2A cells bearing fluorescent aggregates was determined. Ethosuximide
treatment significantly reduced the fraction of transfected cells containing aggregates
by 20 % compared with the vehicle control (Fig. 4c), although neuronal viability as quantified by SYTOX Orange staining was unaffected
by ethosuximide (Additional file 15: Figure S13). Therefore, ethosuximide can antagonise protein aggregation in worm
neurons in vivo and in mammalian neurons in vitro.